This invention relates to methods and compositions for inactivating viruses in biological compositions.
Transmission of viral diseases (e.g., hepatitis A, B, and C, acquired immunodeficiency syndrome, cytomegalovirus infections) by blood or blood products is a significant problem in medicine. The screening of donor blood for viral markers can help reduce the transmission of viruses to recipients, but many screening methods are directed to only a few discrete viruses and are therefore incomplete or less than 100% sensitive. Furthermore, other biological compositions, such as mammalian and hybridoma cell lines, products of cell lines, milk, colostrum, and sperm, can contain infectious viruses as well.
It is therefore important to inactivate viruses contained in donor blood, blood products, or other biological compositions. At the same time, it is desirable to leave the structure and function of valuable constituents, such as red blood cells, platelets, leukocytes, proteins, and polysaccharides, relatively unchanged.
A number of virus inactivating agents have been developed empirically. For example, formalin, beta-propiolactone, and gamma radiation have been used to inactivate viruses. In addition, ethyleneimine monomer has been used to inactivate foot-and-mouth disease virus and binary ethyleneimine (i.e., ethyleneimine monomer generated by a combination of two reagents) has been used to inactivate feline enteric coronavirus.
Many of these agents modify viruses nonspecifically; methods using these agents can therefore be difficult to standardize and apply reproducibly. Furthermore, many of these agents inactivate only some of the viruses present in a given biological composition.
The invention features a method of inactivating a virus in a biological composition; the method includes the steps of (a) contacting the composition with an organic solvent under viral inactivating conditions, where the organic solvent is selected from the group consisting of (i) trialkylphosphates of the formula PO(OR1)(OR2)(OR3), where each of R1, R2, and R3 is, independently, C1-10 alkyl, (ii) ethers of the formula R4xe2x80x94Oxe2x80x94R5, where each of R4 and R5 is, independently, a C1 to C18 alkyl or alkenyl radical which can contain an oxygen or sulfur atom, and (iii) alcohols of the formula R6xe2x80x94OH, where R6 is a C1 to C18 alkyl or alkenyl radical which can contain 1 to 4 oxygen or sulfur atoms, inclusive, in the chain, and which can be substituted by 1 to 4 hydroxy groups, inclusive, and (b) contacting the composition with an ethyleneimine oligomer inactivating agent under viral inactivating conditions, where steps (a) and (b) are performed less than 24 hours apart. Preferably, the inactivating agent is ethyleneimine dimer or ethyleneimine trimer.
A preferred method further includes the step of (c) contacting the composition with a nonionic detergent, such as polyoxyethylene sorbitan monooleate, under viral inactivating conditions, where steps (a), (b), and (c) are performed less than 24 hours apart. Preferred solvents include tri-n-butyl phosphate, ethers of the formula R4xe2x80x94Oxe2x80x94R5, where each of R4 and R5 is, independently, a C1 to C8 alkyl radical, and alcohols of the formula R6xe2x80x94OH, where R6 is a C1 to C8 alkyl radical; a preferred detergent is polyoxyethylene sorbitan monooleate.
Preferably, steps (a), (b), and (c) are performed substantially simultaneously.
The invention also features a composition containing an organic solvent as described above, a non-ionic detergent, and an ethyleneimine oligomer inactivating agent.
The invention further features a method of inactivating a virus in a biological composition; the method includes the steps of (a) contacting the composition with an organic solvent under viral inactivating conditions, where the organic solvent is selected from the group consisting of (i) trialkylphosphates of the formula PO(OR1)(OR2)(OR3), where each of R1, R2, and R3 is, independently, C1-10 alkyl, (ii) ethers of the formula R4xe2x80x94Oxe2x80x94R5, where each of R4 and R5 is, independently, a C1 to C18 alkyl or alkenyl radical which can contain an oxygen or sulfur atom, and (iii) alcohols of the formula R6xe2x80x94OH, where R6 is a C1 to C18 alkyl or alkenyl radical which can contain 1 to 4 oxygen or sulfur atoms, inclusive, in the chain, and which can be substituted by 1 to 4 hydroxy groups, inclusive, and (b) contacting the composition with an inactivating agent under viral inactivating conditions, where the inactivating agent has the formula: 
where each of R1, R2, R3, R4, R6, R7, and R8 is, independently, H or a monovalent hydrocarbon moiety containing between 1 and 4 carbon atoms, inclusive, provided that R1, R2, R3, R4, R6, R7, and R8 cannot all be H; R5 is a divalent hydrocarbon moiety containing between 2 and 4 carbon atoms, inclusive; X is a pharmaceutically acceptable counter-ion; and n is an integer between 1 and 10, inclusive, where steps (a) and (b) are performed less than 24 hours apart.
A preferred method further includes the step of (c) contacting the composition with a nonionic detergent under viral inactivating conditions, where steps (a), (b), and (c) are performed less than 24 hours apart. In another preferred method, steps (a), (b), and (c) are performed substantially simultaneously.
The invention also features a composition including an organic solvent as described above, a non-ionic detergent, and an inactivating agent, where the inactivating agent has the formula: 
where each of R1, R2, R3, R4, R6, R7, and R8 is, independently, H or a monovalent hydrocarbon moiety containing between 1 and 4 carbon atoms, inclusive, provided that R1, R2, R3, R4, R6, R7, and R8 cannot all be H; R5 is a divalent hydrocarbon moiety containing between 2 and 4 carbon atoms, inclusive; X is a pharmaceutically acceptable counter-ion; and n is an integer between 1 and 10, inclusive.
The invention also features a method of inactivating a virus in a biological composition; the method includes the steps of (a) contacting the composition with an organic solvent under viral inactivating conditions, where the organic solvent is selected from the group consisting of (i) trialkylphosphates of the formula PO(OR1)(OR2)(OR3), where each of R1, R2, and R3 is, independently, C1-10 alkyl, (ii) ethers of the formula R4xe2x80x94Oxe2x80x94R5, where each of R4 and R5 is, independently, a C1 to C18 alkyl or alkenyl radical which can contain an oxygen or sulfur atom, and (iii) alcohols of the formula R6xe2x80x94OH, where R6 is a C1 to C18 alkyl or alkenyl radical which can contain 1 to 4 oxygen or sulfur atoms, inclusive, in the chain, and which can be substituted by 1 to 4 hydroxy groups, inclusive, and (b) contacting the composition with an inactivating agent under viral inactivating conditions, where the inactivating agent has the formula
xcfx89-X1xe2x80x94[R1xe2x80x94N+(R2, R3)xe2x80x94]nR4.(X2xe2x88x92)n
where X1 is Cl or Br; R1 is a divalent hydrocarbon moiety containing between 2 and 4 carbon atoms, inclusive; each of R2, R3, and R4 is, independently, H or a monovalent hydrocarbon moiety containing between 1 and 4 carbon atoms, inclusive; X2 is a pharmaceutically acceptable counter-ion; and n is an integer between 2 and 10, inclusive, where steps (a) and (b) are performed less than 24 hours apart. Preferably, R1 is ethylene; R2, R3, and R4 are H; and n is 3 or 4.
A preferred method further includes the step of (c) contacting the composition with a nonionic detergent under viral inactivating conditions, where steps (a), (b), and (c) are performed less than 24 hours apart. In another preferred method, steps (a), (b), and (c) are performed substantially simultaneously.
The invention further features a composition including an organic solvent as described above, a non-ionic detergent, and an inactivating agent, where the inactivating agent has the formula:
xcfx89-X1xe2x80x94[R1xe2x80x94N+(R2, R3)xe2x80x94]nR4.(X2xe2x88x92)n
where X1 is Cl or Br; R1 is a divalent hydrocarbon moiety containing between 2 and 4 carbon atoms, inclusive; each of R2, R3, and R4 is, independently, H or a monovalent hydrocarbon moiety containing between 1 and 4 carbon atoms, inclusive; X2 is a pharmaceutically acceptable counter-ion; and n is an integer between 2 and 10, inclusive.
In addition, the invention features a method of inactivating a virus in a biological composition; the method includes the steps of (a) contacting the composition with an organic solvent under viral inactivating conditions, where the organic solvent is selected from the group consisting of (i) trialkylphosphates of the formula PO(OR1)(OR2)(OR3), where each of R1, R2, and R3 is, independently, C1-10 alkyl, (ii) ethers of the formula R4xe2x80x94Oxe2x80x94R5, where each of R4 and R5 is, independently, a C1 to C18 alkyl or alkenyl radical which can contain an oxygen or sulfur atom, and (iii) alcohols of the formula R6xe2x80x94OH, where R6 is a C1 to C18 alkyl or alkenyl radical which can contain 1 to 4 oxygen or sulfur atoms, inclusive, in the chain, and which can be substituted by 1 to 4 hydroxy groups, inclusive, and (b) contacting the composition with an inactivating agent under viral inactivating conditions, where the inactivating agent has the formula:
xcfx89-X1xe2x80x94CH2CH2xe2x80x94N+H(R1)xe2x80x94[R2xe2x80x94N+(R3, R4)xe2x80x94]nR5.(X2xe2x88x92)n+1
where X1 is Cl or Br; each of R1, R3, R4, and R5 is, independently, H or a monovalent hydrocarbon moiety containing between 1 and 4 carbon atoms, inclusive; R2 is a divalent hydrocarbon moiety containing 3 or 4 carbon atoms; X2 is a pharmaceutically acceptable counter-ion; and n is an integer between 1 and 10, inclusive, where steps (a) and (b) are performed less than 24 hours apart.
A preferred method further includes the step of (c) contacting the composition with a nonionic detergent under viral inactivating conditions, where steps (a), (b), and (c) are performed less than 24 hours apart. In another preferred method, steps (a), (b), and (c) are performed substantially simultaneously.
Finally, the invention features a composition including an organic solvent as described above, a non-ionic detergent, and an inactivating agent, where the inactivating agent has the formula:
xcfx89-X1xe2x80x94CH2CH2xe2x80x94N+H(R1)xe2x80x94[R2xe2x80x94N+(R3, R4)xe2x80x94]nR5.(X2xe2x88x92)n+1
where X1 is Cl or Br; each of R1, R3, R4, and R5 is, independently, H or a monovalent hydrocarbon moiety containing between 1 and 4 carbon atoms, inclusive; R2 is a divalent hydrocarbon moiety containing 3 or 4 carbon atoms; X2 is a pharmaceutically acceptable counter-ion; and n is an integer between 1 and 10, inclusive.
xe2x80x9cINACTINE(trademark) agentsxe2x80x9d refers to compounds of the invention having (1) an aziridino moiety or a halo-hydrocarbon-amino moiety, such as a xcex2-ethyl-amino moiety, and (2) two or more nitrogen atoms separated by hydrocarbon moieties. These compounds are also referred to as xe2x80x9cinactivating agents,xe2x80x9d or xe2x80x9cselective inactivating agents.xe2x80x9d
An inactivating agent has xe2x80x9cselectivityxe2x80x9d for nucleic acids or xe2x80x9cselectivelyxe2x80x9d reacts with nucleic acids if the comparative rate of reaction of the inactivating agent with nucleic acids is greater than the rate of reaction with other biological molecules, e.g., proteins, carbohydrates or lipids.
xe2x80x9cNucleic acidxe2x80x9d refers to both single and double stranded DNA and RNA.
xe2x80x9cBiological compositionxe2x80x9d refers to a composition containing or derived from cells or biopolymers. Biological compositions include, for example, whole blood, red cell concentrates, platelet concentrates, leukocyte concentrates, blood cell proteins, blood plasma, platelet-rich plasma, a plasma concentrate, a precipitate from any fractionation of the plasma, a supernatant from any fractionation of the plasma, blood plasma protein fractions, purified or partially purified blood proteins or other components, serum, semen, mammalian colostrum, milk, saliva, placental extracts, a cryoprecipitate, a cryosupernatant, a cell lysate, mammalian cell culture or culture medium, products of fermentation, ascitic fluid, proteins present in blood cells, and products produced in cell culture by normal or transformed cells (e.g., via recombinant DNA or monoclonal antibody technology). Biological compositions can be cell-free.
xe2x80x9cBiopolymerxe2x80x9d or xe2x80x9cbiological moleculexe2x80x9d refers to any class of organic molecule normally found in living organisms including, for example, nucleic acids, polypeptides, post-translationally modified proteins (e.g., glycoproteins), polysaccharides, and lipids.
xe2x80x9cInactivating,xe2x80x9d xe2x80x9cinactivation,xe2x80x9d or xe2x80x9cinactivate,xe2x80x9d when referring to nucleic acids, means to substantially eliminate the template activity of DNA or RNA, for example, by destroying the ability to replicate, transcribe, or translate a message. When referring to viruses, the term xe2x80x9cinactivatingxe2x80x9d means diminishing or eliminating the number of infectious viral particles measured as a decrease in the infectious titer or number of infectious virus particles per ml. Such a decrease in infectious virus particles is determined by assays well known to a person of ordinary skill in the art.
xe2x80x9cViral inactivating conditionsxe2x80x9d refer to the conditions under which the viral particles are incubated with the inactivating compositions of this invention, including, for example, time of treatment, pH, temperature, salt composition, and concentration of selective inactivating agent, so as to inactivate the viral genome to the desired extent. Viral inactivating conditions are selected from the conditions described below for the selective inactivation of viruses in biological compositions.
xe2x80x9cVirusxe2x80x9d refers to DNA and RNA viruses, viroids, and prions. Viruses include both enveloped and non-enveloped viruses, for example, hepatitis A virus, hepatitis B virus, poxviruses, herpes viruses, adenoviruses, papovaviruses, parvoviruses, reoviruses, orbiviruses, picornaviruses, rotaviruses, alphaviruses, rubivirues, influenza virus, type A and B, flaviviruses, coronaviruses, paramyxoviruses, morbilliviruses, pneumoviruses, rhabdoviruses, lyssaviruses, orthmyxoviruses, bunyaviruses, phleboviruses, nairoviruses, hepadnaviruses, arenaviruses, retroviruses, enteroviruses, rhinoviruses and the filoviruses.
xe2x80x9cEthyleneimine oligomersxe2x80x9d refer to compounds of the formula: 
where n is an integer from 1 to 10, inclusive, and salts thereof. The compounds can be linear or branched.
The methods and compositions of the present inventions provide advantages over other approaches to inactivating viruses in biological compositions. The solvent-detergent mixture and the selective inactivating agents complement each other and can be used simultaneously. The simultaneous treatment of biological compositions with two different inactivating agents provides a relatively fast, inexpensive method for inactivating a wide variety of viruses. Furthermore, since INACTINE(trademark) agents are selective for nucleic acids and solvent-detergent mixtures are selective for the lipid coating of lipid-containing viruses, valuable components of the biological composition, such as proteins and carbohydrates, are left intact.
Other features and advantages of the invention will be apparent from the following description and from the claims.